CN113832042B - Salt-tolerant Saccharomycopsis fibuligera and application thereof in high-salt high-COD sewage treatment - Google Patents

Abstract

The invention belongs to the field of environmental microorganisms, and particularly relates to a salt-tolerant ascochyta tectorial membrane yeast and application thereof in high-salt high-COD sewage treatment. For the problem of poor microbial high-salt tolerance in high-salt and high-COD sewage, the invention provides a strain of salt-tolerant saccharomyces cerevisiae (Saccharomyces fibuligera) which is preserved in Guangdong collection of microorganisms with the preservation number of GDMCC No. 61874. The invention also provides application of the salt-tolerant saccharomycete tectorial membrane yeast in high-salt and high-COD sewage treatment. The salinity tolerance range of the saccharomycete strain of the Fujianfecti provided by the invention is 0-20%, the degradation of COD can be carried out under the conditions that the NaCl content is 3-5% and the initial COD is not less than 15000mg/L, and the effluent can reach the third level of the Integrated wastewater discharge Standard GB 8978-1996.

Description

A salt-tolerant yeast strain of S. forticulata and its application in the treatment of high-salt and high-COD sewage

technical field

The invention belongs to the field of environmental microorganisms, and in particular relates to a strain of salt-tolerant yeast S. forticulata and its application in high-salt and high-COD sewage treatment.

Background technique

Halophilic and halophilic fungi are a type of extreme environmental eukaryotic microorganisms that can grow in a high-salt environment or require a high-salt environment to grow. For a long time, people have always believed that there is no fungus in a high-salt environment. It was not until 1985 that Radewan et al. isolated some salt-loving fungi from Egyptian soil, which were mainly identified as Aspergillus and Penicillium. This confirmed the high-salt environment. There are also fungal flora in low-temperature environments. Since then, some new halophilic and halal-tolerant fungi have been isolated from high-salt environments such as salt lakes and salt fields, or some salt-cured foods. It can grow in an environment with a mass fraction of 3%-30% NaCl; Li Yanhua isolated a strain of Strepospora halophile from a salt field, which can grow in an environment of 5%-30% NaCl; Saritac isolated several strains of fungi from the Dead Sea, respectively Belonging to Penicillium and Aspergillus, they can grow in 20%-25% NaCl environment. However, because halophilic and halophilic fungi grow slowly and lack competitiveness, compared with halophilic archaea and halophilic bacteria, there are still few reports on halophilic and halophilic fungi. Halophilic and halo-tolerant fungi have a unique salt-tolerant mechanism and can often produce special metabolites, which are of great significance for industrial production of antibiotics, organic acids, industrial enzymes, improvement of plant salt and drought resistance, and development of new materials. Therefore, halophilic fungi are a class of microorganisms with great research value.

Usually, the high-salt sewage has a relatively high salt content, >1%, and some are as high as 10%, and because the biodegradability of chemical high-salt and high-COD sewage is extremely weak, the high osmotic pressure and high salinity caused by high salinity The concentration of chloride ions will have a serious impact on the survival, growth and reproduction of most microorganisms to a certain extent. The enzymatic activity of common microorganisms is inhibited in a high-salt environment; therefore, it is key to screen out strains that are tolerant to high-salt environments in activated sludge.

Contents of the invention

The technical problem to be solved by the invention is to deal with the problem that microorganisms in high-salt and high-COD sewage have poor high-salt tolerance.

The technical solution adopted by the present invention to solve the technical problem is: a strain of salt-tolerant Saccharomycopsis fibuligera, the preservation number of which is GDMCC No: 61874. Preserved in the Guangdong Microbial Culture Collection Center, the preservation date is August 18, 2021, and the address is: 5th Floor, Building 59, Compound Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

Wherein, the above-mentioned Saccharomyces spp. has the nucleotide sequence shown in SEQ ID NO:1.

SEQ ID NO: 1 The nucleotide 18S rDNA sequence 1333bp of Saccharomyces forticulata is as follows:

ccgatagtcc ctctaagaag taactatatc agcaaacgct aacagtacta tttagtaggt 60

taaggtctcg ttcgttatcg caattaagca gacaaatcac tccaccaact aagaacggcc 120

atgcaccacc accccacaaaa tcaagaaaga gctctcaatc tgtcaatcct tattgtgtct 180

ggacctggtg agtttccccg tgttgagtca aattaagccg caggctccac tcctggtggt 240

gcccttccgt caattccttt aagtttcagc cttgcgacca tactcccccc agaacccaaa 300

aactttgatt tctcgtaagg tgccgagtga gtcagtaaaa gaacaacacc cgatccctag 360

tcggcatagt ttatggttaa gactacgacg gtatctgatc atcttcgatc ccctaacttt 420

cgttcttgat taatgaaaac gtccttggca aatgctttcg cagtagttag tcttcaataa 480

atccaagaat ttcacctctg acaattgaat actgatgccc ccgaccgtcc ctattaatca 540

ttacgatggt cctagaaacc aacaaaatag aaccatacgt cctatttcat tattccatgc 600

taatatattc gagctaaacg cctgctttga acactctaat tttttcaaag taatagtcct 660

ggatcatatg cagctgagac aagcccaact aacacagaaac caggaggaaa ggctcggctg 720

aaaaccagta ctcgttaaaa aacggaccgg ccagccaagc ccaaagttca actacgagct 780

ttttaactgc aacaacttta atatacgctc ttggagctgg aattaccgcg gctgctggca 840

ccagacttgc cctccaattg ttcctcgtta aggtatttaa attgtactca ttccaattac 900

aagacccgta agggccctgc atcgttatat attgtcacta cctccctgtg tcaggattgg 960

gtaatttgcg cgcctgctgc cttccttgga tgtggtagcc gtttctcagg ctccctctcc 1020

ggaatcgaac ccttattccc cgttacccgt tgaaaccatg gtaggccact atcctaccat 1080

cgaaagttga tagggcagaa atttgaatga accatcgcca gcatgaagcc ttgcgattcg 1140

agaagttat atgaatcacc aaagagcacc gaagtgcatt ggttttttat ctaataaata 1200

catcccttcc aaacagtcgg gatttttagc atgtattagc tctagaatta ccacggttat 1260

ccaagtagta aaggtactat caaataaacg ataactgatt taatgagcca ttcgcagttt 1320

cactgtataa ttg 1333

Wherein, the morphological characteristics of the strain of the above-mentioned Saccharomyces fornicosa yeast are off-white, with a rough surface, irregular colony edges, and radial filaments.

Wherein, the individual shape of the above-mentioned yeast S. fornicata under the optical microscope is: filamentous, 2.8-6.09um wide, with transverse stems and bulging knots.

Wherein, the high-salt range (calculated by NaCl mass) tolerated by the above-mentioned Saccharomyces fornicosa yeast is 30-150 g/L.

Wherein, the range of high salt tolerated by the above-mentioned Saccharomyces fornicosa yeast is 30-50 g/L.

Wherein, the high COD tolerance range of the above-mentioned yeast S. forticulata is 15000-17000 mg/L.

The present invention also provides the application of the salt-tolerant yeast S. forticulata in the treatment of high-salt and high-COD sewage.

The beneficial effects of the present invention are: the salinity tolerance range of the Saccharomyces spp. provided by the present invention is 0%-20%, and it can be used under the conditions of 3-5% NaCl and an initial COD of not less than 15000mg/L. Carry out the degradation of this COD, through continuous detection, find that its effluent can reach the third grade of "Comprehensive Wastewater Discharge Standard" GB8978-1996. The strain XW-1 has extremely high salinity tolerance and can remove COD in a high-salt environment, so this strain has potential research and application value in the salt tolerance mechanism and industrial application.

The yeast strain of Saccharomycopsis fibuligera provided by the present invention was preserved in Guangdong Microorganism Culture Collection Center GDMCC on August 18, 2021, the preservation number is GDMCC No: 61874, and the biological classification is named Saccharomycopsis fibuligera.

Description of drawings

Fig. 1 is the colony morphological characteristics of the salt-tolerant yeast S. forticulata of the present invention.

Fig. 2 is the individual morphological characteristics of the salt-tolerant S. forticulata strain of the present invention.

Fig. 3 is the growth situation of the salt-tolerant S. forticulata strain of the present invention under different salt concentrations.

Fig. 4 shows the COD removal effect of the salt-tolerant yeast strain of S. forticulata of the present invention at a salt concentration of 30 g/L.

Fig. 5 shows the COD removal effect of the salt-tolerant yeast strain of S. forticulata of the present invention at a salt concentration of 50 g/L.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

The invention provides a strain of salt-tolerant Saccharomycopsis fibuligera, the preservation number of which is GDMCC No: 61874. Preserved in the Guangdong Microbial Culture Collection Center, the preservation date is August 16, 2021, and the address is: 5th Floor, Compound Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

The above-mentioned salt-tolerant yeast strain of S. forticulata is isolated from Luzhou Laojiao distiller's grains. The morphological characteristics of the strain on the PDA plate are: off-white, rough surface, irregular colony edges, and radial filaments; individual morphology under the optical microscope It is: filamentous, 2.8-6.09um wide, with horizontal stems and swollen knots; the strain number is XW-1; it has the nucleotide sequence shown in SEQ ID NO:1.

The tolerant high salt range of the present invention is 30-150g/L, and the preferred implementation salt concentration is 30-50g/L, and the high COD range is 15000-17000mg/L.

The present invention also provides the application of the above-mentioned salt-tolerant yeast S. forticulata in the treatment of high-salt and high-COD sewage.

The specific embodiment of the present invention will be further explained below by the examples, but it does not mean that protection scope of the present invention is limited in the scope described in the examples.

Embodiment 1 Isolation and identification of bacterial strains of the present invention

Take 10g of Luzhou Laojiao distiller's grains, add them into a cone containing ^90mL of sterile water, shake at 120r/min at 30°C for 30min, and then dilute in a ^{10 -fold} gradient. Spread 0.1 mL of the bacterial suspension on a PDA plate containing 10% NaCl and incubate at 28°C for 5-7 days. The colony grown on the plate was isolated and purified again by the dilution coating method until a pure culture was obtained, named XW1, and stored in a 30% glycerol tube at -80°C. The colony morphological characteristics of strain XW1 are shown in Figure 1, and the individual morphological characteristics are shown in Figure 2.

Example 2 Determination of Salt-tolerant Growth of Bacterial Strains

Salinity tolerance analysis of the strain XW1, the strains of the strain XW1 were respectively inoculated in the Cultivate in LB plate and liquid, culture upside down at 37°C, observe and record the colony growth in 24 hours as shown in Table 1. Under different salt concentration conditions, the initial OD600 of the strain XW1 was controlled to be consistent. After 72 hours, the OD600 growth was measured to determine whether the strain could tolerate and grow, as shown in Figure 3.

Table 1 Salinity gradient test results of strain XW1

serial number salinity gradient Observations (5d) serial number salinity gradient Observations (5d) 1 0 ++++ 1 0 ++++ 2 1% ++++ 2 10% +++ 3 2% +++ 3 13% ++ 4 3% +++ 4 15% + 5 5% +++ 5 18% + 6 7% +++ 6 20% + 7 9% +++ 7 25% +

Note: "++++" looks good, "+++" looks good; "++" looks average, "+" can grow, and "-'" can't.

Example 3 High-salt and high-COD sewage removal effect experiment

(1) Preparation of wet cells: the XW1 cell fermentation broth was obtained after the strain XW1 was activated and fermented.

(2) Monitoring of salt-tolerant COD removal performance: Pour into the laboratory moving bed bioreactor (Moving Bed Biofilm Reactor, MBBR), the laboratory distribution water influent NH ₄ -N concentration 50mg/L, COD concentration 15000-17000mg/ L, the concentration of sodium chloride is 30-50mg/L, and the daily detection indicators include pH, NH ₄ -N, NO ₂ -N, NO ₃ -N, TP, TN and regularly analyze the salt-tolerant COD removal performance of the strain XW1 under the MBBR process .

(3) After adding salt-tolerant bacteria, the verification test of the strain XW1 lasted for one month, the sodium chloride in the influent rose from 30g/L to 50g/L, and the COD concentration in the effluent of the MBBR reaction tank in the laboratory was basically about 600mg/L. Aqueous ammonia nitrogen is completely degraded.

(4) The application of strain XW1 in the treatment of high-salt sewage. The concentration of sodium chloride in the influent is 30g/L, and the initial COD concentration is 17000mg/L. After 18 days of detection and research, it is found that the effluent on the 13th day can reach the sewage discharge standard Level three; as shown in Figure 4.

(5) The application of strain XW1 in high-salt sewage treatment, the influent sodium chloride concentration is 50g/L, and the initial COD concentration is 15000mg/L. After 20 days of detection and research, it is found that the effluent water can reach the sewage discharge standard on the 18th day Level three; as shown in Figure 5.

comparative example

Treatment of high-salt high-COD sewage and operating conditions are the same as in Example 3, except that Candida rugosa (Candida rugosa) is added during the treatment process. Since the salt concentration is still 30-50g/L, the high COD of the effluent remains unchanged and slightly increases. Considering that Candida rugosa does not have the function of removing high COD with salt tolerance, the treatment effect is not good, and the operation fails.

The culture conditions and biotransformation process of the strain XW1 can be applied to the treatment of other high-salt wastewater besides high-salt wastewater. As can be seen from the foregoing embodiments, the present invention has the following advantages compared with the prior art:

1. The bacterial strain XW1 cultivated by the present invention has a high concentration of flora and good activity, can quickly become a dominant bacterium in an open system, and can exert strain-specific functions;

2. The high-salt tolerance of the strain XW1 reaches above 15% NaCl, and has strong living activity and tolerance;

3. It can play a biochemical treatment function in high-salt and high-COD wastewater, and is suitable for the treatment of various high-salt and high-COD wastewater;

4. It can grow under the condition that COD is greater than 15000mg/L. Compared with the Rhodococcus erythropolis reported in the patent CN 111117913 A, the highest COD load of its influent is only 12000mg/L; the fungal strain XW1 of the present invention is its The tolerance to high concentrations of COD is better than that of Rhodococcus erythropolis.

5. The bacterial strain XW1 cultivated by the present invention can solve the problem of high-salt and high-COD waste water in the third grade of "Comprehensive Wastewater Discharge Standard" GB8978-1996.

sequence listing

<110> Luzhou Laojiao Co., Ltd.

Nanjing High-Tech University Biotechnology Research Institute Co., Ltd.

Luzhou Pinchuang Technology Co., Ltd.

<120> A salt-tolerant yeast strain of S. forticulata and its application in the treatment of high-salt and high-COD sewage

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1333

<212>DNA

<213> Artificial Sequence

<400> 1

ccgatagtcc ctctaagaag taactatatc agcaaacgct aacagtacta tttagtaggt 60

taaggtctcg ttcgttatcg caattaagca gacaaatcac tccaccaact aagaacggcc 120

atgcaccacc accccacaaaa tcaagaaaga gctctcaatc tgtcaatcct tattgtgtct 180

ggacctggtg agtttccccg tgttgagtca aattaagccg caggctccac tcctggtggt 240

gcccttccgt caattccttt aagtttcagc cttgcgacca tactcccccc agaacccaaa 300

aactttgatt tctcgtaagg tgccgagtga gtcagtaaaa gaacaacacc cgatccctag 360

tcggcatagt ttatggttaa gactacgacg gtatctgatc atcttcgatc ccctaacttt 420

cgttcttgat taatgaaaac gtccttggca aatgctttcg cagtagttag tcttcaataa 480

atccaagaat ttcacctctg acaattgaat actgatgccc ccgaccgtcc ctattaatca 540

ttacgatggt cctagaaacc aacaaaatag aaccatacgt cctatttcat tattccatgc 600

taatatattc gagctaaacg cctgctttga acactctaat tttttcaaag taatagtcct 660

ggatcatatg cagctgagac aagcccaact aacacagaaac caggaggaaa ggctcggctg 720

aaaaccagta ctcgttaaaa aacggaccgg ccagccaagc ccaaagttca actacgagct 780

ttttaactgc aacaacttta atatacgctc ttggagctgg aattaccgcg gctgctggca 840

ccagacttgc cctccaattg ttcctcgtta aggtatttaa attgtactca ttccaattac 900

aagacccgta agggccctgc atcgttatat attgtcacta cctccctgtg tcaggattgg 960

gtaatttgcg cgcctgctgc cttccttgga tgtggtagcc gtttctcagg ctccctctcc 1020

ggaatcgaac ccttattccc cgttacccgt tgaaaccatg gtaggccact atcctaccat 1080

cgaaagttga tagggcagaa atttgaatga accatcgcca gcatgaagcc ttgcgattcg 1140

agaagttat atgaatcacc aaagagcacc gaagtgcatt ggttttttat ctaataaata 1200

catcccttcc aaacagtcgg gatttttagc atgtattagc tctagaatta ccacggttat 1260

ccaagtagta aaggtactat caaataaacg ataactgatt taatgagcca ttcgcagttt 1320

cactgtataa ttg 1333

Claims (2)

1. A strain of salt-tolerant Saccharopolyspora fibuligera is characterized in that: the salt-tolerant saccharomyces cerevisiae fibuligera is deposited with GDMCC No. 61874 and preserved in Guangdong collection of microorganisms with the preservation date 2021, 8 and 16 days.

2. The use of the salt-tolerant Saccharomyces cerevisiae of claim 1 for the treatment of wastewater with NaCl concentrations of 30-50g/L and high COD ranges of 15000-17000mg/L.

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